Abstract

Resistance to transforming Growth factor (TGF) β-mediated tumor suppression represents a major step in melanoma aggressiveness. At the cell surface, TGF-β assembles a complex of transmembrane receptor serine/threonine kinases (types I and II) and induces transphosphorylation and activation of the type I receptor (TβR-I, ALK5) by the type II receptor kinase (TβR-II). The activated type I receptor phosphorylates the downstream effectors Smad2 and Smad3, which then associate with a common Smad4, and these activated complexes translocate into the nucleus, where they regulate transcription of target genes. Smad2 and Smad3 activities mediate TGFβ growth inhibitory effects by 1) downregulation of c-myc, CDC25A and Id family members; and 2) upregulation of p15 and p21 cyclin-dependent kinase inhibitors. In order to investigate whether high levels of c-myc could play a role in TGFβ resistance, we analyzed c-myc expression in 13 human melanoma cell lines. 6 melanoma cell lines demonstrated high levels of the protein c-myc, a repressor of p21 and p15 expression. Interestingly, these cell lines also had low levels of p21, raising the possibility that high c-myc levels were maintaining low levels of this cyclin-dependent kinase inhibitor in these lines. We were then interested to know whether low levels of Smad2 or/and Smad3 could potentially be responsible for high c-myc levels in the cells. Our preliminary data shows that while all 13 melanoma cell lines had comparable levels of Smad2 expressed, 7 out of the 13 had lower expression of Smad3. Interestingly, 6 of the 7 with low Smad3 expression were the lines with the highest levels of c-myc and the lowest levels of p21. We therefore suggest that these cell lines may have selected for a lower Smad3 expression as a way to render themselves insensitive to TGFβ-mediated growth suppression. In order to directly address the role of high c-myc levels in the resistance to TGFβ-mediated growth inhibition, we are knocking down the expression of c-myc in the resistant melanoma cell lines with high levels of the protein. We are analyzing these cells to determine if they are now less resistant to TGFβ. On the other hand, we are over-expressing c-myc in TGFβ sensitive melanoma cell lines (Radial Growth Phase melanomas) with low levels of c-myc, with the expectation that these melanoma lines will become more resistant to TGFβ. We will correlate these results to the expression levels of p15 and p21. In order to determine whether reduction of Smad3 expression could represent a mechanism leading to high c-myc expression, we will over-express Smad3 in the melanoma cell lines with low Smad3 expression and concomitant high c-myc and low p21 levels and determine whether we can restore sensitivity to TGFβ-mediated growth inhibition. Taken together our results allude to a novel mechanism used by melanoma cells which may involve utilizing c-myc to evade TGFβ mediated suppression.